Study on a Mechanism of Improving MaAPX1 Protein Activity by Mutating Methionine to Lysine

Ascorbate peroxidases (APXs) are key components of the ascorbate–glytathione cycle, which plays an important role in removing excess reactive oxygen species (ROS) in plants. Herein, MaAPX1 was verified as being involved in the ripening and senescence of banana fruit, exhibiting responsiveness to the accumulation of ROS and the oxidation of proteins. Site-directed mutation was applied to explore the mechanism of MaAPX1 activity changes. We found that the 32-site cysteine (Cys, C) served as a potential S-nitrosylation site. The mutant MaAPX1C32S activity was decreased significantly when Cys32 was mutated to serine (Ser, S). Intriguingly, the neighboring conserved 36-site methionine (Met, M), which is adjacent to Cys32, displayed an enzyme activity that was approximately five times higher than that of the wild-type MaAPX1 when mutated to lysine (Lys, K). Utilizing LC-MS/MS spectroscopy coupled with stopped-flow analysis showed that the enhanced MaAPX1M36K activity might be due to the increased S-nitrosylation level of Cys32 and the promotion of intermediate (compound I, the first intermediate product of the reaction of APX with H2O2) production. Molecular docking simulations showed that the S-N bond between Cys32 and Lys36 in MaAPX1M36K might have a function in protecting the thiol of Cys32 from oxidation. MaAPX1M36K, a promising mutant, possesses immense potential for improving the antioxidant capabilities of APX in the realm of bioengineering technology research.

APX activity is regulated by multiple post-translational modifications, such as Snitrosylation, carbonylation, glutathionylation, metal nitrosylation, and Cys oxidation and nitration [16].The cys32 in APX is highly conserved and present (100%) in the cytoplasm of APX [17,18].Cys32 is part of the pocket that binds to ASC (ascorbate), and its nitrosylation can promote APX activity [19].In the case of ROS accumulation, the activity of A. toxicaria APX can be inhibited by carbonylation modification in seed-drying processes [20].The oxidized cysteine in APX can be reduced by the NADPH-Trx (nicotinamide adenine dinucleotide phosphate-thioredoxin) system to restore protein activity [21], and APX activity is inhibited when the thiol group of cysteine is affected by reduced glytathione [17].Furthermore, APX, as a typical heme protein, has an inhibitory effect on its activity when its heme subunit undergoes metal chelation [22,23].As a pivotal antioxidant protein capable of efficiently neutralizing reactive oxygen species, augmenting the activity of APX is crucial for enhancing plant resilience against environmental stresses.To date, in addition to the documented enhancement of APX activity through S-nitrosylation modification, there is an imperative need to elucidate and establish novel methods that can potentiate the enzymatic activity of APX.
Bananas (Musa sp.cv.Brazil) are one of the most economically important fruits and subject to a swift process of ripening and senescence once harvested.APX is a pivotal component of the antioxidant enzyme system.Our preliminary study revealed that mutating Met36 into glutamine (Gln, Q) in MaAPX1 (Ma02_p20860.1)significantly decreased APX activity by hindering the formation of compound I [24].The transformation of Met36 into various amino acids elicited diverse responses in enzyme activity.In our present study, when the Met36 in MaAPX1 was mutated into lysine (K), the enzyme activity improved, showing enzyme activity that was about five times higher than that of the wild-type MaAPX1.To elucidate the underlying mechanism of this activity enhancement in MaAPX1 M36K , we employed a multi-faceted approach, including LC-MS/MS (liquid chromatography-mass spectrometry), spectral analysis, and molecular docking.MaAPX1 M36K , as a new mutant material, offers immense potential in augmenting the antioxidant function of APX in the realm of bioengineering technology research.

Plant Materials and Treatments
Bananas (Musa sp.cv.Brazil) with a maturity of 70-80% were harvested from Nansha District, Guangzhou, Guangdong Province, China, and transported to a laboratory within 2 h.Fruits with uniform size and color were selected and soaked in 0.1% Sportak ® (Prochloraz, Bayer, Leverkusen, Germany) for 3 min and then allowed to air-dry.
Three treatments were set up in this experiment, including a control, an ethylene treatment, and a 1-MCP treatment.Sixty dried bananas were placed in an 80 L sealed plastic box to ensure air circulation inside.For the ethylene treatment group, the sealed box was filled with 40 mL of ethylene; for the 1-MCP (1-Methylcyclopropene, ethylene receptor inhibitor) treatment group, 1-MCP was placed in the sealed box to achieve a final concentration of 1 ppm.All the covers of the sealed boxes were removed for ventilation after 24 h.Each treatment had 3 replicates.Based on the hardness and color changes during banana storage, samples were taken to determine the relevant physiological indicators.Partial samples were stored in a −80 • C refrigerator for RNA extraction.

Measurement of Physiological Parameters
Banana peel color was measured with a Konica Minolta CR-400 colorimeter (Konica Minolta Co., Ltd., Tokyo, Japan) in the CIEL*a*b mode.L* indicated the lightness ranging from black to white, and hue angle (h*) referred to a color wheel, which was red at an angle of 0 • , yellow at 90 • , green at 180 • , and blue at 270 • , in accordance with the method outlined by McGuire [25].
The H 2 O 2 content and protein carbonylation level in the banana peels were determined using a hydrogen peroxide kit and protein carbonyl content kit (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China), respectively.The content was expressed as µmol g −1 FW.
The steps taken for total APX enzyme extraction and activity measurement were as follows: One point five grams of fresh banana peel was ground in liquid nitrogen and dissolved in 3 mL of pre-cooled 50 mM potassium phosphate buffer (pH7.0) to extract APX for 10 min.The mixture was centrifuged at 4 • C at 10,000 rpm, and the supernatant was collected to measure the enzyme activity.Reaction system: 1 mL 50 mM potassium phosphate buffer (pH 7.0), 0.4 mL of 0.3 mM EDTA-Na 2 , and 1 mL of 0.9 mM VC were mixed, and 0.1 mL of enzyme solution was added to initiate the reaction.We recorded the changes in the OD value of the reaction solution at 290 nm for 2 min and repeated this process three times.APX activity (µmol VC mg −1 min −1 FW) = ∆A × Vt × v/(FW × Vs × 2.8 × t); 'Vt'-total volume of enzyme solution, mL; 'FW'-fresh weight of the sample, mg; 'Vs'-volume of enzyme solution taken during measurement, mL); 'v'-volume of reaction solution, mL; '∆A'-the absorbance value changes of OD 290 within 60 s; 't'-Reaction time, min; and '2.8'-the millimolar extinction coefficient of VC.

Plasmid Construction and Gene Expression
The coding sequences of MaAPX1 were PCR-amplified from banana complementary DNA.Total RNA was extracted from banana peel tissues using the hot borate method [26].DNA-free RNA was used as the template for reverse-transcription PCR.The gene-specific primers used for gene cloning and gene expression are listed in Table S2.The PCR products were sub-cloned into the pMD20-T vector (TaKaRa) and then transformed into E. coli DH5α (TaKaRa) in accordance with the manufacturer's protocol.The sequences were verified via further cloning and resequencing.The resulting sequences were deposited in Banana Genome Hub https://banana-genome-hub.southgreen.fr/(accessed on 9 July 2024) with the accession number GSMUA_Achr5P07280_001 (MaAPX1).The alignments were carried out using ClustalX (version 1.83).The qRT-PCR reactions for MaAPX1 were carried out using the ABI 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) with SYBR Green Real-Time PCR Master Mix (TOYOBO Co., Ltd., Osaka, Japan).The conditions of the reactions were set with reference to the work by Xiao et al. [27] with the reference gene MaActin-3 [28].Three independent biological replicate experiments were performed in this analysis.

Site-Directed Mutagenesis of Cys32 and Met36 Residues
The following site-directed mutagenesis of Cys32 or Met36 was performed: mutations of Cys32 into serine (S) and Met36 into lysine (K) were carried out as described previously [24].The full-length MaAPX1 mutants were sub-cloned into DH5α and verified through DNA sequencing.The oligonucleotide sequences of responding mutation sites in MaAPX1 for RT-PCR are shown in Table S2.

Purification of Recombinant Proteins
The sequences encoding MaAPX1 or MaAPX1 mutants were inserted into pET-28a (+) vector (Novagen, Irene, South Africa).The His-fusion proteins were induced and expressed in E.coli BL21 (DE3) strain (TransGen Biotech, Beijing, China).The recombinant proteins were then purified with nickel-nitrilotriacetic acid agarose (Qiagen, Hilden, Germany), following the manufacturer's instructions.The results of SDS-PAGE of purified expressed recombinant MaAPX1-His and the mutants from E. coli BL21 are shown in Figure S2.

Enzymatic Activity of MaAPXs Measurement
MaAPX activity was measured according to the method reported by Yang et al. [19], with some modifications.The protein concentrations of MaAPXs were adjusted to 0.1 mg/mL with 50 mM sodium phosphate (pH 7.0).As typically carried out, 500 µL of the reconstitu-tion products was used for the assay by adding sodium ascorbate and hydrogen peroxide to final concentrations of 0.45 mM and 1 Mm, respectively.After initiating the reaction by adding hydrogen peroxide, the samples were immediately analyzed with UVmini-1240 by measuring the absorbance at A290 every 15 s for 2 min.APX activity (µM AsA min −1 µg −1 ) =∆A × 1000 × 1000/(C × Vs × 2.8 × t); 'C'-concentration of the sample, mg/mL; 'Vs'-volume of enzyme solution, µL; '∆A'-the absorbance value changes at OD290 within 1 min; 't'-reaction time, min; and '2.8'-the millimolar extinction coefficient.
To investigate the effect of in vitro S-nitrosation on protein activity, the solvent of purified MaAPX1-His was replaced with HEN buffer (250 mM Hepes, pH 7.7; 1 mM EDTA-Na 2 ; 0.2 mM neocuproine) using an ultrafiltration centrifuge tube.Then, the protein was added to a final concentration of 750 µM GSNO (Sigma Aldrich, St. Louis, MO, USA, Cat no.: N4148) and incubated in a dark room.After one hour, the solvent of the protein was replaced with 50 mM sodium phosphate (pH 7.0), and then the protein activity was measured.

Mass-Spectrometric Analysis of S-Nitrosylation Level of Proteins
Mass-spectrometric analysis of S-nitrosylation level of proteins was conducted by Shenzhen Wininnovate Biotechnology Limited Company (Shenzhen, China).
Protein digestion.A total of 50 µg of purified MaAPX1-His was denatured with 8 M urea and then diluted to 1 M urea with 50 mM NH 4 HCO 3 .The protein dilution material was digested with trypsin (1:50 enzyme/protein ratio) at 37 • C for 16-18 h and acidified with 1% TFA (Trifluoroacetic Acid).The peptides were lyophilized.
Nano LC-ESI-MS/MS analysis.The lyophilized peptides were re-suspended in 2% acetonitrile containing 0.1% formic acid, 4 µL aliquots of which were loaded into a ChromXP C18 (3 µm, 120 Å) trap column.Online chromatography separation was performed using the Ekspert nanoLC 415 system (SCIEX, Concord, ON, Canada).The trapping-anddesalting procedure was carried out at a flow rate of 4 µL/min for 5 min with 100% solvent A (water/acetonitrile/formic acid (98/2/0.1%).Then, an elution gradient of 8-38% solvent B (2/98/0.1%) in 40 min was used on an analytical column (75 µm × 15 cm C18-3 µm 120 Å, ChromXP, Eksigent, Dublin, CA, USA).IDA (information-dependent acquisition) mass spectrum techniques were used to acquire tandem MS data using a Triple TOF 6600 tandem mass spectrometer (Sciex, Concord, ON, Canada) fitted with a Nano spray III ion source.Data were acquired using an ion spray voltage of 2.4 kV, curtain gas of 35 PSI, nebulizer gas of 12 PSI, and an interface heater temperature of 150 • C. The MS was operated with TOF-MS scans.For IDA, survey scans were acquired in 250 ms, and up to 40 product ion scans (50 ms) were collected if a threshold of 260 cps with a charge state of 2-4 was exceeded.The rolling collision energy setting was applied to all precursor ions for collision-induced dissociation.Dynamic exclusion was set for 16 s.
Database searching.The MS/MS data were analyzed for protein modification (Carboxamidomethylation (+57.02) of cysteine, oxidation (+15.99) of methionine, acetylation (+42.01) of Protein N-term (deamidation (+0.98) of asparagine and S-nitrosylation (+28.99) of cysteine were allowed as variable modifications), and quantification using PEAKS Studio version 8.5 (BSI, San Jose, CA, USA) against protein sequence.The threshold used for peptide identification was a peptide for which −10lgP > 20 and that had a PTM A score > 500.Mass accuracy of ±20 ppm was used for precursor ions, and 0.5 Da was used for product ions.Enzyme specificity was limited to trypsin, with at least one tryptic (K-or R-containing) terminus required per peptide and up to two miscleavages being allowed.Cysteine carboxamidomethylation was specified as a static modification; oxidations of methionine residue and the appropriate PTM were allowed as variable modifications.

Spectroscopic Analysis and Stopped-Flow Analysis
According to the method employed by Hugo et al. [29], UV-visible spectra of Asc-free MaAPX1 and mutant MaAPX1 M36K (1 µg/µL) samples were recorded at 25 • C in the presence or absence of an equimolar H 2 O 2 concentration in potassium phosphate buffer (50 mM, pH 7.0).
Rapid-acquisition spectra and absorbance time courses were obtained following the recording of the Asc-free MaAPX1 and mutant MaAPX1 M36K enzymes before and after H 2 O 2 addition by mixing equal amounts (2 µg/µL each) in potassium phosphate buffer (50 mM, pH 7.0) containing diethylenetriaminepentaacetic acid (DTPA) (0.1 mM) at 10 • C, reaching a final concentration of 1 µg/µL of reagents.Absorbance was recorded with an Applied Photophysics 9CD stopped-flow spectrofluorometer equipped with a rapidscanning diode array.Formation of compound I species followed, occurring at a Soret peak of 409 nm for MaAPX1 and 408 nm for MaAPX1 M36K .

Molecular Docking Experiment
The 3D structures of MaAPX1 and its mutants (MaAPX1 M36K ) were generated by using Swiss-Model, using the crystal structure of soybean APX (PDB: 1V0H, 84.53% sequence identity) as an initial template [30].Models of ligands (heme) were constructed using eLBOW, Phoenix, by using smile strings of ligands via the AM1/QM optimization method.The model of MaAPX1 and ligands were prepared using Autodock tool.In this case, Cys32 and Met36 (or Lys36) were set as flexible residues.

Statistical Analysis
Data were expressed as the means ± SE (standard error) of three biological replicates.Differences among different treatments were compared using SPSS version 7.5 (SPSS, Inc., Chicago, IL, USA).Different letters in each figure indicate significant differences (p < 0.05) between different treatments at the same storage time.

Ripening Characteristics and Redox Status of Harvested Banana Fruit during Ripening and Senescence
Bananas are a typical climacteric fruit, characterized by a pre-climacteric phase followed by a respiration peak, subsequently becoming soft and yellow.As shown in Figure 1A,B, the control fruits underwent a yellowing process after 14 days; in contrast, the ethylene (C 2 H 4 )-treated fruits exhibited yellowing on the 4nd day, and the 1-MCP treated fruits took 33 days to turn yellow.The control fruits experienced a significant reduction in hug angle, declining from 119.1 to 110.5 on the 14nd day, and this decline was accompanied with yellowing.The emergence of conspicuous spots and browning of the peel occurred on the 22nd day, with a decreased hug angle of 93.8.This indicated that the fruit ripened and underwent greater senescence as the storage time increased.Ethylene promoted yellowing of the banana peel, and the hug angle of the banana peel decreased to 105.4 on the fourth day; spots appeared on the 8nd day, with a decrease in the hug angle amounting to 92.6, which is much lower than that for the control group (p < 0.05).In contrast, for the 1-MCP-treated fruit, the hug angle decreased to 108.1 after 26 days of storage, and peel browning occurred after 33 days of storage.These results indicate that ethylene promoted the ripening and senescence of bananas, while 1-MCP delayed ripening and senescence.
H 2 O 2 is a by-product produced by organisms during aerobic respiration [31].A low concentration of H 2 O 2 acts as an important signal, while a high concentration of H 2 O 2 causes oxidative damage to biological macromolecules.Figure 1C shows an accumulation of H 2 O 2 in banana peels along with different ripening stages.C 2 H 4 treatment accelerated the accumulation of H 2 O 2 , while 1-MCP treatment delayed the accumulation of H 2 O 2 .Figure 1D shows the changes in APX activity in the banana peels, which are basically consistent with the accumulation of H 2 O 2 .APX activity in the C 2 H 4 -and 1-MCP-treated bananas increased with the accumulation of H 2 O 2 during storage, and there was a strong correlation between APX activity and H 2 O 2 content during fruit ripening and senescence (p < 0.05) (Table S1).These results indicate that APX might be a pivotal enzyme, responsible for the elimination of H 2 O 2 during banana ripening and senescence.
causes oxidative damage to biological macromolecules.Figure 1C shows an accumulation of H2O2 in banana peels along with different ripening stages.C2H4 treatment accelerated the accumulation of H2O2, while 1-MCP treatment delayed the accumulation of H2O2. Figure 1D shows the changes in APX activity in the banana peels, which are basically consistent with the accumulation of H2O2.APX activity in the C2H4-and 1-MCP-treated bananas increased with the accumulation of H2O2 during storage, and there was a strong correlation between APX activity and H2O2 content during fruit ripening and senescence (p < 0.05) (Table S1).These results indicate that APX might be a pivotal enzyme, responsible for the elimination of H2O2 during banana ripening and senescence.Most biological macromolecules (nucleic acids, proteins, lipids) in living organisms are the target substances for ROS oxidation, and these biological macromolecules are prone to structural and functional damage and accelerate the process of aging after oxidation [32].Carbonylation modification occurs after proteins are oxidized, and it is accompanied by the formation of sulfonic acid and sulfone substances, which have a great impact on the function of a protein [33].Protein carbonylation levels are commonly used to evaluate the degree of protein oxidation.In the present study, protein carbonylation Most biological macromolecules (nucleic acids, proteins, lipids) in living organisms are the target substances for ROS oxidation, and these biological macromolecules are prone to structural and functional damage and accelerate the process of aging after oxidation [32].Carbonylation modification occurs after proteins are oxidized, and it is accompanied by the formation of sulfonic acid and sulfone substances, which have a great impact on the function of a protein [33].Protein carbonylation levels are commonly used to evaluate the degree of protein oxidation.In the present study, protein carbonylation levels increased gradually as the storage time increased (Figure 1E), and it was observed that the C 2 H 4 and 1-MCP treatments played roles in promoting and inhibiting protein carbonylation, respectively.These findings underscore the significance of ROS accumulation in triggering oxidative damage to biological macromolecules, thereby serving as a crucial factor in accelerating the senescence of fruits.

Expression Profiles of MaAPX1 Gene during Banana Ripening and Senescence
To further verify the function of MaAPX1 in banana ripening and senescence, we conducted a comprehensive analysis of MaAPX1 gene expression in banana peels during storage (Figure 2).The relative expression level of the MaAPX1 gene, under both the control and C 2 H 4 treatments, displayed a trend of upregulation followed by a subsequent downregulation trend as storage progressed (Figure 2A,B).In stark contrast, the MaAPX1 expression in the bananas treated with 1-MCP showed consistent downregulation throughout the entire storage phase (Figure 2C), indicating that MaAPX1 likely played a pivotal role in orchestrating the regulatory mechanisms governing fruit ripening and senescence.
in triggering oxidative damage to biological macromolecules, thereby serving as a crucial factor in accelerating the senescence of fruits.

Expression Profiles of MaAPX1 Gene during Banana Ripening and Senescence
To further verify the function of MaAPX1 in banana ripening and senescence, we conducted a comprehensive analysis of MaAPX1 gene expression in banana peels during storage (Figure 2).The relative expression level of the MaAPX1 gene, under both the control and C2H4 treatments, displayed a trend of upregulation followed by a subsequent downregulation trend as storage progressed (Figure 2A,B).In stark contrast, the MaAPX1 expression in the bananas treated with 1-MCP showed consistent downregulation throughout the entire storage phase (Figure 2C), indicating that MaAPX1 likely played a pivotal role in orchestrating the regulatory mechanisms governing fruit ripening and senescence.

The MaAPX1 Activity Is Promoted by S-Nitrosylation of Cys32
The function of cAPX (cytoplasmic APX) is regulated by multiple PTMs (post-translational modifications), among which S-nitrosylation is known to play an important role in regulating cAPX functions.APX1 was identified as a putative S-nitrosylation protein through nitrosoproteomic studies [34,35].It has been reported that AtAPX1 enzymatic activity can be enhanced by S-nitrosylation at Cys32 [19].In our present study, the activity of MaAPX1 recombinant protein improved by nearly 20% after treatment with the NO doner GSNO (Figure 3A), and we identified Cys32 as the S-nitrosylated residue in MaAPX1 recombinant protein via LC-MS/MS (Figure 3B), indicating that MaAPX1 is one of the substrates of S-nitrosylation whose enzymatic activity is positively regulated by S-nitrosylation.

The MaAPX1 Activity Is Promoted by S-Nitrosylation of Cys32
The function of cAPX (cytoplasmic APX) is regulated by multiple PTMs (post-translational modifications), among which S-nitrosylation is known to play an important role in regulating cAPX functions.APX1 was identified as a putative S-nitrosylation protein through nitrosoproteomic studies [34,35].It has been reported that AtAPX1 enzymatic activity can be enhanced by S-nitrosylation at Cys32 [19].In our present study, the activity of MaAPX1 recombinant protein improved by nearly 20% after treatment with the NO doner GSNO (Figure 3A), and we identified Cys32 as the S-nitrosylated residue in MaAPX1 recombinant protein via LC-MS/MS (Figure 3B), indicating that MaAPX1 is one of the substrates of S-nitrosylation whose enzymatic activity is positively regulated by Snitrosylation.Purified MaAPX1-His recombinant protein was treated with GSNO for 1 h and then assayed for its enzymatic activities (A).Mass-spectrometric analysis of the tryptic fragments of GSNO-untreated MaAPX1-His recombinant protein, in which Cys32 was identified as an S-nitrosylated residue (the mass charge ratio (m/z) of Cys in the mass spectrum was 103.01, and it increased (+28.99) to 132.00 after S-nitrosylation).The fragment ions y5 and y6 were not detected due to their low SNRs (signal-to-noise ratios).The occurrence of S-nitrosylation in C and deamidation in N led to an increase in m/z for y7, y8, y9, and y10 (+28.99 + 0.98) that indicated we could still be sure that C was S-nitrosylated and N was deamidated.The associated mass ions are given in red in the table.(B).Each datapoint represents a mean ± standard error (n = 3).The values with different letters indicate that each sampling from different treatment groups is significantly different (p < 0.05).Mass-spectrometric analysis of the tryptic fragments of GSNO-untreated MaAPX1-His recombinant protein, in which Cys32 was identified as an S-nitrosylated residue (the mass charge ratio (m/z) of Cys in the mass spectrum was 103.01, and it increased (+28.99) to 132.00 after S-nitrosylation).The fragment ions y5 and y6 were not detected due to their low SNRs (signal-to-noise ratios).The occurrence of S-nitrosylation in C and deamidation in N led to an increase in m/z for y7, y8, y9, and y10 (+28.99 + 0.98) that indicated we could still be sure that C was S-nitrosylated and N was deamidated.The associated mass ions are given in red in the table.(B).Each datapoint represents a mean ± standard error (n = 3).The values with different letters indicate that each sampling from different treatment groups is significantly different (p < 0.05).

Mutant MaAPX1 M36K Activity Might Be Enhanced by Improving the S-Nitrosylation Level of Cys32
If continuous fragmentation of y5 and y6 occurs, it can be clearly seen that the corresponding m/z of y5 ion will increase by 28.99 after S-nitrosylation in Cys, and the corresponding y6 ion m/z will increase by 0.98 after deamidation in Asn (N).
There are two cysteine residues in MaAPX1, namely, Cys32 and Cys167, of which Cys32 is the candidate S-nitrosylation residue in MaAPX1 identified via LC-MS/MS (Figure 3B) combined with the prediction made using GPS-GNO software [36].The Cys32 and Met36 in APXs are highly conserved in various species (Figure S1).There was a typical active site, APLM 36 LRLAWHSA, at the N-terminus end, accompanied by a heme ligand of DIVALSGGHTL in MaAPX1, signifying the pivotal position of Met36 in the enzymatic core.As shown in Figure 4A, the substitution of Cys32 with a Ser residue (termed MaAPX1 C32S ) led to a more than 60% reduction in MaAPX1 activity, while the substitution of Met36 with Lys (termed MaAPX1 M36K ) led to an increase in enzymatic activity that was about five times higher than that for MaAPX1.These results indicate that Cys32 and Met36 are important for the function of MaAPX1.In addition, the relative S-nitrosylation level of Cys32 in the mutant MaAPX1 M36K recombinant protein was much higher than that of wild MaAPX1, as determined using LC-MS/MS, indicating that MaAPX1 M36K was more prone to S-nitrosylation, and MaAPX1 M36K might be S-nitrosylated during the process of prokaryotic expression or protein purification (Figure 4B).Combined with the improvement of enzymatic activity as well as the S-nitrosylation level on Cys32 for MaAPX1 M36K , it can be inferred that S-nitrosylation at Cys32 may play a pivotal role in promoting the activity of MaAPX1 M36K .
active site, APLM36LRLAWHSA, at the N-terminus end, accompanied by a heme ligand of DIVALSGGHTL in MaAPX1, signifying the pivotal position of Met36 in the enzymatic core.As shown in Figure 4A, the substitution of Cys32 with a Ser residue (termed MaAPX1 C32S ) led to a more than 60% reduction in MaAPX1 activity, while the substitution of Met36 with Lys (termed MaAPX1 M36K ) led to an increase in enzymatic activity that was about five times higher than that for MaAPX1.These results indicate that Cys32 and Met36 are important for the function of MaAPX1.In addition, the relative S-nitrosylation level of Cys32 in the mutant MaAPX1 M36K recombinant protein was much higher than that of wild MaAPX1, as determined using LC-MS/MS, indicating that MaAPX1 M36K was more prone to S-nitrosylation , and MaAPX1 M36K might be S-nitrosylated during the process of prokaryotic expression or protein purification (Figure 4B).Combined with the improvement of enzymatic activity as well as the S-nitrosylation level on Cys32 for MaAPX1 M36K , it can be inferred that S-nitrosylation at Cys32 may play a pivotal role in promoting the activity of MaAPX1 M36K .

Visible Spectrum and Stopped-Flow Analysis of MaAPX1 and Mutant MaAPX1 M36K Reaction with H 2 O 2
In our previous study, we verified that the enzymatic activity of MaAPX1 is related to the formation of compound R, either the heme ring or an amino acid side chain) [24].For further exploration of the mechanism of MaAPX1 M36K activity improvement, optical spectra of MaAPX1 and the mutant MaAPX1 M36K (from 350 to 650 nm) were recorded before and after the addition of an equimolar concentration of H 2 O 2 to assay the spectral shifts associated with the formation of a compound-I-like product (Figure 5A,B).In the resting state, MaAPX1 and the mutant MaAPX1 M36K exhibited different Soret peaks at 409 nm and 408 nm (Figure 5A,B), respectively, which indicated that there was some structural perturbation in the heme microenvironment as the result of amino acid replacement [29].As shown in Figure 5A, the Soret peak of MaAPX1 was shifted from the resting state of 409 nm to 410 nm after reaction with H 2 O 2 , with the declined two humps situated at 539 and 574 nm.The Soret peak of MaAPX1 M36K shifted from 408 nm to 414 nm after reaction with H 2 O 2 , with two rising humps at 539 and 574 nm (Figure 5B).Spectral shifts are indicative of compound-Ilike formation.
the mechanism of MaAPX1 M36K activity improvement, optical spectra of MaAPX1 and the mutant MaAPX1 M36K (from 350 to 650 nm) were recorded before and after the addition of an equimolar concentration of H2O2 to assay the spectral shifts associated with the formation of a compound-I-like product (Figure 5A,B).In the resting state, MaAPX1 and the mutant MaAPX1 M36K exhibited different Soret peaks at 409 nm and 408 nm (Figure 5A,B), respectively, which indicated that there was some structural perturbation in the heme microenvironment as the result of amino acid replacement [29].As shown in Figure 5A, the Soret peak of MaAPX1 was shifted from the resting state of 409 nm to 410 nm after reaction with H2O2, with the declined two humps situated at 539 and 574 nm.The Soret peak of MaAPX1 M36K shifted from 408 nm to 414 nm after reaction with H2O2, with two rising humps at 539 and 574 nm (Figure 5B).Spectral shifts are indicative of compound-I-like formation.Given the rapid generation of the compound-I-like product, rapid kinetics analysis via the stopped-flow method at 10 °C was performed to visualize the classical compound I in MaAPX1 and the mutant MaAPX1 M36K .The maximal absorption (Soret peak) in the ferric state of heme peroxidases dropped in intensity when the enzyme reacted with H2O2 to generate compound I; a decrease in absorbance at the Soret peak is expected after H2O2 reaction [37][38][39].In our present study, a swift drop in absorbance for MaAPX1 M36K (Soret peak at 408 nm) was observed when it was mixed with H2O2, indicating the formation of Given the rapid generation of the compound-I-like product, rapid kinetics analysis via the stopped-flow method at 10 • C was performed to visualize the classical compound I in MaAPX1 and the mutant MaAPX1 M36K .The maximal absorption (Soret peak) in the ferric state of heme peroxidases dropped in intensity when the enzyme reacted with H 2 O 2 to generate compound I; a decrease in absorbance at the Soret peak is expected after H 2 O 2 reaction [37][38][39].In our present study, a swift drop in absorbance for MaAPX1 M36K (Soret peak at 408 nm) was observed when it was mixed with H 2 O 2 , indicating the formation of the Fe-IV = O -P •+ radical (P •+ , porphyrin-centered radical) of compound I (Figure 5D).Contrastingly, there was no decrease in absorbance for MaAPX1 (Figure 5C), indicating that the improvement in MaAPX1 M36K activity might also stem from the expedited generation of compound I.
Furthermore, the mutant MaAPX1 M36K exhibited increased absorption in the Soret region with high spin state variation, indicating that the activity of iron increased and that the iron became more prone to engaging in reactions with the other substance.Conversely, a reduced spin state exhibited greater stability and a diminished tendency to interact with other substances.This underscores the pivotal role of the spin state in compound I, which significantly impacts the subsequent reaction of compound I (or compound II, APX (Fe 4+ = O) R • + ASA → APX (Fe 4+ = O)R (compound II) + monodehydroascorbate) with ascorbate.Accordingly, the elevated iron spin in compound I coupled with the swift formation rate of compound I emerge as critical determinants of the elevated enzymatic activity exhibited by MaAPX1 M36K .

Molecular Docking Study of MaAPX1
The pocket surrounding Cys32 emerges as a vital heme-binding site, crucial in enhancing the functionality of ascorbate peroxidase.Based on the high sequence identity between MaAPX1 and the soybean APX, the predicted model (depicted in orange) was generally overlayed with the template model (depicted in blue), with minor variations observed in the flexible loop's structural conformation (Figure 6A).The alignment between MaAPX1 and its mutant variant, MaAPX1 M36K , exhibits a striking 100% sequence identity.This high degree of conservation suggests that the mutant MaAPX1 M36K can comfortably integrate into the overall protein structure without perturbing its essential components, including the area around the Met36 residue (Figure S3A,B).Furthermore, Cys32 is deemed an indispensable amino acid residue in terms of the catalytic activity of cAPX, and the pocket it anchors further underlines its crucial contribution to APX activity, highlighting the significance of this region in the enzyme's overall functionality.Watanabe et al. [40] reported that the Cys32 was the core of the heme-binding site and catalytic center in human peroxiredoxin (Prx).Therefore, it can be hypothesized that Cys32 plays an important role in ligand recognition or even catalytic activity.As depicted in Figure 6B, Cys32 was observed to be a promising site for heme recognition or binding.It may either identify heme and facilitate its transfer to the core binding site or bind to heme firmly and display catalytic prowess in ascorbic acid metabolism.In docking simulations focused on the heme's central binding site, expanding the grid range indicated the emergence of a secondary binding locale surrounding Cys32, further validating its potential for heme interaction.This is consistent with the reduction in the enzymatic activity of MaAPX1 C32S (Figure 4A).Within this prospective binding locale, Cys32 orchestrates the formation of transition metal thiolate complexes with heme's iron, effectively supplanting histidine as the heme's binding nucleus.Moreover, three polar or positively charged residues, Arg166 (Arginine), His168 (histidine), and Arg171, were located on the opposite side of the heme molecule, whose positively charged region can act as a proton supplier.As shown in Figure 6C, the docking positions of both heme and ascorbic acid also showed that, when attached to the heme molecule, the two hydroxyl groups from ascorbic acid were capable of interacting with Arg166 and Arg171.The geometry of the Cys32 pocket fulfills the condition as an APX catalytic site.

The Lys36 Residue of MaAPX1 M36K Might Induce S-Nitrosylation of Cys32 via S-N Bonds to Enhance Enzymatic Activity
We have verified that the occurrence of S-nitroslysation at the thiol group of Cys32 significantly enhanced the activity of MaAPX1.However, unfortunately, in molecular docking simulations, the nitroso group positioned on the cysteine side chain cannot be explicitly modeled.Nonetheless, the reversibility of S-nitroslysation through de-nitrosylation processes in the presence of metal ions and under photolytic conditions ensures that the Snitroslysation at Cys32 does not impede heme binding.As depicted in Figure 7, during the docking simulations in mutant MaAPX1 M36K , the unrestricted torsion of Cys32 and Lys36 led to conformational alterations in Cys32, subsequently forming an S-N bond with Lys36, with the heme molecule binding to a place to that for MaAPX1.While the precise role of this S-N bond remains elusive, we postulate that Lys36 may induce the S-nitroslysation of Cys32, thereby shielding the thiol group from potential oxidative reactions and contributing to an increase in enzymatic activity.

The Lys36 Residue of MaAPX1 M36K Might Induce S-Nitrosylation of Cys32 via S-N Bonds to Enhance Enzymatic Activity
We have verified that the occurrence of S-nitroslysation at the thiol group of Cys32 significantly enhanced the activity of MaAPX1.However, unfortunately, in molecular docking simulations, the nitroso group positioned on the cysteine side chain cannot be explicitly modeled.Nonetheless, the reversibility of S-nitroslysation through de-nitrosylation processes in the presence of metal ions and under photolytic conditions ensures that the S-nitroslysation at Cys32 does not impede heme binding.As depicted in Figure 7, during the docking simulations in mutant MaAPX1 M36K , the unrestricted torsion of Cys32 and Lys36 led to conformational alterations in Cys32, subsequently forming an S-N bond with Lys36, with the heme molecule binding to a place to that for MaAPX1.While the precise role of this S-N bond remains elusive, we postulate that Lys36 may induce the S-nitroslysation of Cys32, thereby shielding the thiol group from potential oxidative reactions and contributing to an increase in enzymatic activity.Furthermore, it has been reported that the oxidation of Cys-SH can be divided into reversible oxidation and irreversible oxidation [41].Figure 8 shows reversible cysteine oxidation taking place in hydrogen peroxide.In the case of irreversible oxidation, the thiol group undergoes oxidation to form R-SO3H due to persistent exposure to H2O2 without the presence of a deoxidizer.Conversely, in the reversible oxidation pathway, Lys36 in the mutant MaAPX1 M36K variant, possessing a positive charge and situated in close proximity to Cys32, may serve as a source of protons, functioning as a deoxidizer for R-SOH (depicted by the red dotted arrow).This prompt reduction of R-SOH back to R-SH ensures the timely protection of Cys32 from undergoing irreversible oxidation.Furthermore, it has been reported that the oxidation of Cys-SH can be divided into reversible oxidation and irreversible oxidation [41].Figure 8 shows reversible cysteine oxidation taking place in hydrogen peroxide.In the case of irreversible oxidation, the thiol group undergoes oxidation to form R-SO 3 H due to persistent exposure to H 2 O 2 without the presence of a deoxidizer.Conversely, in the reversible oxidation pathway, Lys36 in the mutant MaAPX1 M36K variant, possessing a positive charge and situated in close proximity to Cys32, may serve as a source of protons, functioning as a deoxidizer for R-SOH (depicted by the red dotted arrow).This prompt reduction of R-SOH back to R-SH ensures the timely protection of Cys32 from undergoing irreversible oxidation.
the mutant MaAPX1 variant, possessing a positive charge and situated in close proximity to Cys32, may serve as a source of protons, functioning as a deoxidizer for R-SOH (depicted by the red dotted arrow).This prompt reduction of R-SOH back to R-SH ensures the timely protection of Cys32 from undergoing irreversible oxidation.Figure 8. Reversible cysteine oxidation in hydrogen peroxide.After being exposed to H2O2, thiol groups of Cys are initially oxidized into a sulfenic acid form, which is highly unstable, and readily react with another proximal thiol to form adisulfide bonds.In a cellular environment, disulfide bonds can be reduced to the thiol form by the thioredoxinor the GSH/glutaredoxin systems (red solid arrow).In the presence of more H2O2, sulfenic acid can be further oxidized to sulfinic or sulfonic acid, which, in general, is an irreversible modification that cannot be repaired within cells.

Conclusions
APX, a pivotal constituent of the ascorbic acid-glutathione cycle, holds significant importance in governing H2O2 metabolism and stands as a chief modulator of cellular redox equilibrium in plants.In the present study, MaAPX1 was shown to be involved in the process of banana ripening and senescence.The Met36 in MaAPX1, which is close to Cys32, is a crucial residue and may act as a switch regulating the enzymatic activity of After being exposed to H 2 O 2 , thiol groups of Cys are initially oxidized into a sulfenic acid form, which is highly unstable, and readily react with another proximal thiol to form adisulfide bonds.In a cellular environment, disulfide bonds can be reduced to the thiol form by the thioredoxinor the GSH/glutaredoxin systems (red solid arrow).In the presence of more H 2 O 2 , sulfenic acid can be further oxidized to sulfinic or sulfonic acid, which, in general, is an irreversible modification that cannot be repaired within cells.

Conclusions
APX, a pivotal constituent of the ascorbic acid-glutathione cycle, holds significant importance in governing H 2 O 2 metabolism and stands as a chief modulator of cellular redox equilibrium in plants.In the present study, MaAPX1 was shown to be involved in the process of banana ripening and senescence.The Met36 in MaAPX1, which is close to Cys32, is a crucial residue and may act as a switch regulating the enzymatic activity of MaAPX1.Intriguingly, we observed a remarkable enhancement in enzymatic activity when Met36 was substituted with Lys.Drawing upon the insights gained from LC-MS/MS, spectroscopy, stopped-flow analysis, and the comprehensive molecular docking study, we attribute the augmentation of MaAPX1 M36K activity to multiple factors.These include the elevation in the S-nitrosylation levels of Cys32, the accelerated formation of high-spin iron intermediates (compound I), and the plausible establishment of a S-N bond between Cys32 and Lys36 in MaAPX1 M36K .This bond formation safeguards the thiol group of Cys32 from oxidation, thereby bolstering the enzymatic efficacy of MaAPX1.Emerging as a novel mutant material with the potential to enhance protein activity, MaAPX1 M36K presents a valuable resource for further inquiries into the functional aspects of APX and its potential applications in the realm of biotechnology.

Figure 1 .
Figure 1.Ripening characteristics and redox statuses of harvested bananas during ripening and senescence.Changes in the ripening of the banana peels with different treatments (A), hug angle (B), H2O2 content (C), APX activity (D), and protein carbonylation (E).Each data point represents a mean ± standard error (n = 3).The values with different letters refer to each sampling from the same storage time and are significantly different (p < 0.05).

Figure 1 .
Figure 1.Ripening characteristics and redox statuses of harvested bananas during ripening and senescence.Changes in the ripening of the banana peels with different treatments (A), hug angle (B), H 2 O 2 content (C), APX activity (D), and protein carbonylation (E).Each data point represents a mean ± standard error (n = 3).The values with different letters refer to each sampling from the same storage time and are significantly different (p < 0.05).

Figure 2 .
Figure 2. Changes in the relative expression of MaAPX1 in different treatments.Control (A), C2H4 treatment (B), and 1-MCP treatment (C).The relative gene expression levels of MaAPX1 on different storage days, wherein storage day 0 was chosen as the reference, and the gene expression level on

Figure 2 .
Figure 2. Changes in the relative expression of MaAPX1 in different treatments.Control (A), C 2 H 4 treatment (B), and 1-MCP treatment (C).The relative gene expression levels of MaAPX1 on different storage days, wherein storage day 0 was chosen as the reference, and the gene expression level on day 0 was set to 1.Each datapoint represents a mean ± standard error (n = 3).The values with different letters (a, b, c, etc.) in each group indicate that each sample taken at different storage times is significantly different (p < 0.05).

Figure 3 .
Figure 3. S-nitrosylation of MaAPX1-His recombinant protein.GSNO (S-Nitrosoglutathione, NO doner) enhances the enzymatic activity of MaAPX1-His recombinant protein.Purified MaAPX1-His recombinant protein was treated with GSNO for 1 h and then assayed for its enzymatic activities (A).Mass-spectrometric analysis of the tryptic fragments of GSNO-untreated MaAPX1-His recombinant protein, in which Cys32 was identified as an S-nitrosylated residue (the mass charge ratio (m/z) of Cys in the mass spectrum was 103.01, and it increased (+28.99) to 132.00 after S-nitrosylation).The fragment ions y5 and y6 were not detected due to their low SNRs (signal-to-noise ratios).The occurrence of S-nitrosylation in C and deamidation in N led to an increase in m/z for y7, y8, y9, and y10 (+28.99 + 0.98) that indicated we could still be sure that C was S-nitrosylated and N was deamidated.The associated mass ions are given in red in the table.(B).Each datapoint represents a mean ± standard error (n = 3).The values with different letters indicate that each sampling from different treatment groups is significantly different (p < 0.05).

Figure 3 .
Figure 3. S-nitrosylation of MaAPX1-His recombinant protein.GSNO (S-Nitrosoglutathione, NO doner) enhances the enzymatic activity of MaAPX1-His recombinant protein.Purified MaAPX1-His recombinant protein was treated with GSNO for 1 h and then assayed for its enzymatic activities (A).Mass-spectrometric analysis of the tryptic fragments of GSNO-untreated MaAPX1-His recombinant protein, in which Cys32 was identified as an S-nitrosylated residue (the mass charge ratio (m/z) of Cys in the mass spectrum was 103.01, and it increased (+28.99) to 132.00 after S-nitrosylation).The fragment ions y5 and y6 were not detected due to their low SNRs (signal-to-noise ratios).The occurrence of S-nitrosylation in C and deamidation in N led to an increase in m/z for y7, y8, y9, and y10 (+28.99 + 0.98) that indicated we could still be sure that C was S-nitrosylated and N was deamidated.The associated mass ions are given in red in the table.(B).Each datapoint represents a mean ± standard error (n = 3).The values with different letters indicate that each sampling from different treatment groups is significantly different (p < 0.05).

Figure 4 .
Figure 4. Analysis of the enzymatic activity of MaAPX1-His and the mutants' recombinant proteins via spectrophotometry (A).S-nitrosylation levels of Cys32 in MaAPX1-His and MaAPX1 M36K -His recombinant proteins (B).Mass-spectrometric analysis of the tryptic fragments of GSNO-untreated MaAPX1 M36K -His recombinant protein, in which Cys32 was identified as an S-nitrosylated residue (C).Each datapoint represents a mean ± standard error (n = 3).The values with different letters indicate that each sampling from different treatment groups is significantly different (p < 0.05).

Figure 4 .
Figure 4. Analysis of the enzymatic activity of MaAPX1-His and the mutants' recombinant proteins via spectrophotometry (A).S-nitrosylation levels of Cys32 in MaAPX1-His and MaAPX1 M36K -His recombinant proteins (B).Mass-spectrometric analysis of the tryptic fragments of GSNO-untreated MaAPX1 M36K -His recombinant protein, in which Cys32 was identified as an S-nitrosylated residue (C).Each datapoint represents a mean ± standard error (n = 3).The values with different letters indicate that each sampling from different treatment groups is significantly different (p < 0.05).

Figure 5 .
Figure 5. Spectroscopic analysis of the MaAPX1-His and MaAPX1 M36K -His recombinant proteins compound I and compound I-like intermediates.Absorption spectra of MaAPX1-His (2 µM) (A) and MaAPX1 M36K -His (2 µM) (B) before (solid line) and after (dashed line) equimolar H2O2 addition (2 µM).Spectra from 500 to 650 nm for MaAPX1-His and MaAPX1 M36K -His.Arrows indicate the Soret peak of the resting enzyme before and after H2O2 reaction.Spectral shifts of MaAPX1 M36K -His are indicative of compound-I-like formation.MaAPX1-His (5 µM) (C) or MaAPX1 M36K -His (5 µM) (D) was mixed with H2O2 (dashed line) or left alone (solid line) (5 µM) at 10 °C.The decrease in the absorbance at the Soret peak is indicative of compound I generation.

Figure 5 .
Figure 5. Spectroscopic analysis of the MaAPX1-His and MaAPX1 M36K -His recombinant proteins compound I and compound I-like intermediates.Absorption spectra of MaAPX1-His (2 µM) (A) and MaAPX1 M36K -His (2 µM) (B) before (solid line) and after (dashed line) equimolar H 2 O 2 addition (2 µM).Spectra from 500 to 650 nm for MaAPX1-His and MaAPX1 M36K -His.Arrows indicate the Soret peak of the resting enzyme before and after H 2 O 2 reaction.Spectral shifts of MaAPX1 M36K -His are indicative of compound-I-like formation.MaAPX1-His (5 µM) (C) or MaAPX1 M36K -His (5 µM) (D) was mixed with H 2 O 2 (dashed line) or left alone (solid line) (5 µM) at 10 • C. The decrease in the absorbance at the Soret peak is indicative of compound I generation.

Figure 6 .
Figure 6.Molecular docking study of MaAPX1.Structure of predicted model (orange) and template (PDB: 1V0H) (A).Docking position of heme at the Cys32 binding site of MaAPX1, superimposed with heme at the heme binding site (pink) (B).Docking position of heme and ascorbic acid at the Cys32 binding site of MaAPX1 (C).

Figure 6 .
Figure 6.Molecular docking study of MaAPX1.Structure of predicted model (orange) and template (PDB: 1V0H) (A).Docking position of heme at the Cys32 binding site of MaAPX1, superimposed with heme at the heme binding site (pink) (B).Docking position of heme and ascorbic acid at the Cys32 binding site of MaAPX1 (C).024, 13, x FOR PEER REVIEW 13 of 16

Figure 7 .
Figure 7. Docking of heme with APX1 M36K , in which an interaction between Cys32 and Lys36 can be observed.

Figure 7 .
Figure 7. Docking of heme with APX1 M36K , in which an interaction between Cys32 and Lys36 can be observed.